Ovine GRF

ABSTRACT

A synthetic peptide is extremely potent in stimulating the release of pituitary GH in mammals, particularly in sheep, since it is the replicate of the native (hormone) releasing factor of the sheep hypothalamus. It contains the following sequence: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu. This peptide or a biologically active fragment thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered therapeutically to mammals and may be used diagnostically. The peptide is particularly useful in stimulating the release of GH so as to accelerate growth in warm-blooded non-human animals, particularly sheep, and/or to improve aquiculture.

This invention was made with Government support under grants HD-09690and AM-18811 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

The present invention relates to a peptide having influence on thefunction of the pituitary gland in humans and other mammals. Inparticular, the present invention is directed to a peptide whichpromotes the release of growth hormone by the pituitary gland.

BACKGROUND OF THE INVENTION

Since the early 1950's, physiologists and clinicians have recognizedthat the hypothalamus of the brain controls all the secretory functionsof the adenohypophysis. This control is neurohumoral, with specializedneurosecretory neurons in the hypothalamus producing specialpolypeptides, the effect and role of each of which is to trigger acutelyand chronically the secretion of each pituitary hormone.

An inhibitory factor was earlier characterized in the form ofhypothalamic somatostatin which inhibits, at the pituitary level, thesecretion of growth hormone. In 1982, a corresponding hypothalamicreleasing factor for pituitary growth hormone or somatotropin wasisolated from a human islet cell tumor, purified, characterized andsynthesized. When tested, it was found to promote the release of growthhormone(GH) by the pituitary. This peptide has the sequence:

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂.Human hypothalamic growth hormone releasing factor (hGRF) has now beenfound to have the same structure. Bohlen et. al. Biochem. and Biophs.Res. Comm., 114, 3, pp. 930-936 (1983).

SUMMARY OF THE INVENTION

A 44-residue polypeptide has now been isolated from purified extracts ofovine hypothalami and characterized. It is found to have the amino acidformula:H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Iyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH₂.It is believed to be and is hereinafter referred to as oGRF (for ovinegrowth hormone releasing factor) and will also be termed ovinesomatocrinin. This peptide can be used to promote growth of warm-bloodedanimals, particularly sheep, of cold-blooded animals and in aquiculture.It may also be used to increase milk production in ewes in order toprovide milk for making specialty cheeses.

Pharmaceutical compositions in accordance with the invention includeoGRF, an analog thereof or a biologically active fragment thereof, or anontoxic salt of any of the foregoing, dispersed in a pharmaceuticallyor veterinarily acceptable liquid or solid carrier. Such pharmaceuticalcompositions can be used in clinical medicine, both human andveterinary, in acute or chronic administration for diagnostic ortherapeutic purposes. Moreover, they can be used to accelerate thegrowth of muscle mass in sheep or other animals.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The nomenclature used to define the peptides is that specified bySchroder & Lubke, "The Peptides", Academic Press (1965), wherein inaccordance with conventional representation the amino group at theN-terminal appears to the left and the carboxyl group at the C-terminalto the right. Where the amino acid residue has isomeric forms, it is theL-form of the amino acid that is represented unless otherwise expresslyindicated.

The invention provides synthetic oGRF peptides having the followingformula:H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-Ywherein Y is OH or NH₂. Also included are biologically active fragmentswhere Y can be either OH or NH₂.

The peptides can be synthesized by any suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation, by classical solution couplings, or by theemployment of recently developed recombinant DNA techniques. Forexample, the techniques of exclusively solid-phase synthesis are setforth in the textbook 37 Solid-Phase Peptide Synthesis", Stewart &Young, Freeman & Co., San Francisco, 1969, and are exemplified by thedisclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978. The fragmentcondensation method of synthesis is exemplified in U.S. Pat. No.3,972,859 (Aug. 3, 1976). Other available syntheses are exemplified byU.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925(Jan. 28, 1975). Production of the synthetic peptides using recombinantDNA techniques will likely be used to satisfy large-scale commercialrequirements.

Synthesis by the use of recombinant DNA techniques, for purposes of thisapplication, should be understood to include the suitable employment ofa structural gene coding for a form of oGRF. The synthetic oGRF may beobtained by transforming a microorganism using an expression vectorincluding a promoter and operator together with such structural gene andcausing such transformed microorganism to express oGRF. A non-humananimal may also be used to produce oGRF by gene-farming using such astructural gene and the general techniques set forth in U.S. Pat. No.4,276,282 issued June 30, 1981 or using microinjection of embryos asdescribed in W083/01783 published 26 May 1983 and W082/04443 published23 December 1982. The synthetic oGRF may also be produced directly inthe animal for which accelerated growth is intended by the techniquesdescribed in the two WO publications.

Common to coupling-type syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an alpha-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group, followed by the selectiveremoval of the alpha-amino protecting group to allow subsequent reactionto take place at that location. Accordingly, it is common that, as astep in the synthesis, an intermediate compound is produced whichincludes each of the amino acid residues located in its desired sequencein the peptide chain with side-chain protecting groups linked to theappropriate residues.

Also considered to be within the scope of the present invention areintermediates of the formula: X¹-Tyr(X²)-Ala-Asp(X³)-Ala-Ile-Phe-Thr(X⁴)-Asn-Ser(X⁵)Tyr(X²)-Arg(X⁶)-Lys(X⁷)-Ile-Leu-Gly-Gln-Leu-Ser(X⁵)-Ala-Arg(X⁶)-Lys(X⁷)-Leu-Leu-Gln-Asp(X³)-Ile-Met-Asn-Arg(X⁶)-Gln-Gln-Gly-Glu(X³)-Arg(X⁶)-Asn-Gln-Glu(X³)-Gln-Gly-Ala-Lys(X⁷)-Val-Arg(X⁶)-Leu-X⁸wherein: X¹ is either hydrogen or an α-amino protecting group. Theα-amino protecting groups contemplated by X¹ are those known to beuseful in the art of step-wise synthesis of polypeptides. Among theclasses of α-amino protecting groups covered by X¹ are (1) acyl-typeprotecting groups, such as formyl, trifluoroacetyl, phthalyl,toluenesulfonyl(Tos), benzensulfonyl, nitrophenylsulfenyl,tritylsulfenyl, o-nitrophenoxyacetyl, chloroacetyl, acetyl, andγ-chlorobutyryl; (2) aromatic urethan-type protecting groups, such asbenzyloxycarbonyl(Z) and substituted Z, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphaticurethan protecting groups, such as t-butyloxycarbonyl (BOC),diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;(5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6)alkyl-type protecting groups, such as triphenylmethyl (trityl),benzyl;(7) trialkylsilane groups, such as trimethylsilane. The preferredα-amino protecting group is BOC.

X² is a protecting group for the phenolic hydroxyl group of Tyr selectedfrom the group consisting of tetrahydropyranyl, tert-butyl, trityl, Bzl,CBZ, 4Br-CBZ and 2,6-dichlorobenzyl. The preferred protecting group is2,6-dichlorobenzyl. X² can be hydrogen which means that there is noprotecting group on the hydroxyl group.

X³ is hydrogen or an ester-forming protecting group for the carboxylgroup of Asp or Glu and is selected from the group consisting of Bzl,2,6-dichlorobenzyl, methyl and ethyl.

X⁴ and X⁵ are protecting groups for the hydroxyl group of Thr and Serand are selected from the group consisting of acetyl, benzoyl,tert-butyl, trityl, tetrahydropyranyl, Bzl, 2,6-dichlorobenzyl and CBZ.The preferred protecting group is Bzl. X⁴ and/or X⁵ can be hydrogen,which means there is no protecting group on the hydroxyl group.

X⁶ is a protecting group for the guanidino group of Arg selected fromthe group consisting of nitro, Tos, CBZ, adamantyloxycarbonyl, and BOC,or is hydrogen;

X⁷ is hydrogen or a protecting group for the side chain aminosubstituent of Lys. Illustrative of suitable side chain amino protectinggroups are 2-chlorobenzyloxycarbonyl (2-Cl-Z), Tos, CBZ,t-amyloxycarbonyl and BOC.

The selection of a side chain amino protecting group is not criticalexcept that it must be one which is not removed during deprotection ofthe α-amino groups during the synthesis. Hence, the α-amino protectinggroup and the side chain amino protecting group cannot be the same.

Optionally the side chain amido group of Gln and/or Asn can be suitablyprotected as with xanthyl (Xan).

X⁸ is selected from the class consisting of OH, OCH₃, esters, amides,hydrazides, --0--CH₂ -resin support and --NH-resin support, with thegroups other than OH and amides being broadly considered as protectinggroups.

In the formula for the intermediate, at least one of X¹, X², X³, X⁴, X⁵,X⁶, X⁷, and X⁸ is a protecting group.

In selecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following rules are followed: (a) theprotecting group should be stable to the reagent and under the reactionconditions selected for removing the αamino protecting group at eachstep of the synthesis, (b) the protecting group should retain itsprotecting properties and not be split off under coupling conditions,and (c) the side chain protecting group should be removable, upon thecompletion of the synthesis containing the desired amino acid sequence,under reaction conditions that will not alter the peptide chain.

The peptides are preferably prepared using solid phase synthesis, suchas that described by Merrifield, J. Am. Chem. Soc., 85, p 2149 (1963),although other equivalent chemical syntheses known in the art can alsobe used as previously mentioned. Solid-phase synthesis is commenced fromthe C-terminal end of the peptide by coupling a protected α-amino acidto a suitable resin. Such a starting material can be prepared byattaching α-amino-protected Leu by an ester linkage to achloromethylated resin or a hydroxymethyl resin, or by an amide bond toa BHA resin or MBHA resin. The preparation of the hydroxymethyl resin isdescribed by Bodansky et al., Chem. Ind. (London) 38, 1597-98 (1966).Chloromethylated resins are commercially available from Bio RadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., "Solid PhasePeptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan α-carboxamide at the C-terminal.

Leu protected by BOC is coupled to the chloromethylated resin accordingto the procedure of Monahan and Gilon, Biopolymer 12, pp 2513-19, 1973when, for example, it is desired to synthesize the 44-amino acid peptidewith free carboxy terminal. Following the coupling of BOC-Leu, theα-amino protecting group is removed, as by using trifluoroaceticacid(TFA) in methylene chloride, TFA alone or HCl in dioxane. Thedeprotection is carried out at a temperature between about 0° C and roomtemperature. Other standard cleaving reagents and conditions for removalof specific α-amino protecting groups may be used as described inSchroder & Lubke, "The Peptides", 1 pp 72-75 (Academic Press 1965).

After removal of the α-amino protecting group of Leu, the remainingα-amino- and side chain-protected amino acids are coupled step-wise inthe desired order to obtain the intermediate compound definedhereinbefore, or as an alternative to adding each amino acid separatelyin the synthesis, some of them may be coupled to one another prior toaddition to the solid phase reactor. The selection of an appropriatecoupling reagent is within the skill of the art. Particularly suitableas a coupling reagent is N,N'-dicyclohexyl carbodiimide (DCCI).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are: (1) carbodiimides, such as N,N'-diisopropylcarbodiimide, N-N'-dicyclohexylcarbodiimide(DCCI); (2) cyanamides suchas N,N'-dibenzylcyanamide; (3) keteimines; (4) isoxazolium salts, suchas N-ethyl-5-phenyl isoxazolium-3'-sulfonate; (5) monocyclicnitrogen-containing heterocyclic amides of aromatic character containingone through four nitrogens in the ring, such as imidazolides,pyrazolides, and 1,2,4-triazolides. Specific heterocyclic amides thatare useful include N,N'-carbonyl diimidazole,N,N'-carbonyl-di-1,2,4-triazole; (6) alkoxylated acetylene, such asethoxyacetylene; (7) reagents which form a mixed anhydride with thecarboxyl moiety of the amino acid, such as ethylchloroformate andisobutylchloroformate and (8) reagents which form an active ester withthe carboxyl moiety of the amino acid, such as nitrogen-containingheterocyclic compounds having a hydroxy group on one ring nitrogen, e.g.N-hydroxyphthalimide, N-hydroxysuccinimide and1-hydroxybenzotriazole(HOBT). Other activating reagents and their use inpeptide coupling are described by Schroder & Lubke supra, in Chapter IIIand by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide(DMF):CH₂ Cl₂ (1:1)or in DMF or CH₂ Cl₂ alone. In cases where incomplete coupling occurred,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis ismonitored by the ninhydrin reaction, as described by E. Kaiser et al.,Anal. Biochem. 34, 595 (1970).

After the desired amino acid sequence has been completed, theintermediate peptide is removed from the resin support by treatment witha reagent, such as liquid hydrogen fluoride, which not only cleaves thepeptide from the resin but also cleaves all remaining side chainprotecting groups X², X³, X⁴, X⁵, X⁶, X⁷ and X⁸ and the α-aminoprotecting group X¹, to obtain the peptide.

As an alternative route, the intermediate peptide may be separated fromthe resin support by alcoholysis after which the recovered C-terminalalkyl ester is converted to the acid by hydrolysis. Any side chainprotecting groups may then be cleaved as previously described or byother known procedures, such as catalytic reduction (e.g. Pd on BaSO₄).When using hydrogen fluoride for cleaving, anisole and methylethylsulfide are included in the reaction vessel for scavenging.

The following Example sets forth the preferred method for synthesizingoGRF by the solid-phase technique. It will of course be appreciated thatthe synthesis of a correspondingly shorter peptide fragment is effectedin the same manner by merely eliminating the requisite number of aminoacids at either end of the chain; however, it is presently felt thatbiologically active fragments should contain the indicated sequence atthe N-terminal.

EXAMPLE I

The synthesis of oGRF(1-44) amide having the formula:

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH₂is conducted in a stepwise manner using a Beckman 990 PeptideSynthesizer and an MBHA resin. Coupling of BOC-Leu to the resin isperformed by the general procedure set forth in U.S. Pat. No. 4,292,313,and it results in the substitution of about 0.2-0.6 mmol Leu per gram ofresin depending on the substitution of the MHBA resin used. All solventsthat are used are carefully degassed by sparging with an inert gas,preferably helium, to insure the absence of oxygen that mightundesirably oxidize the sulfur of the Met residue.

After deprotection and neutralization, the peptide chain is builtstep-by-step on the resin. Deprotection, neutralization and addition ofeach amino acid is performed in general accordance with the procedureset forth in detail in Guillemin et al. U.S. Pat. No. 3,904,594. Thecouplings are specifically carried out as set out in the followingschedule.

    ______________________________________                                        SCHEDULE                                                                                                      MIX                                                                           TIMES                                         STEP   REAGENTS AND OPERATIONS  MIN.                                          ______________________________________                                         1     CH.sub.2 Cl.sub.2 (2 times)                                                                            0.5                                            2     50% trifluoroacetic acid (TFA) +                                                                       0.5                                                  5% 1,2-ethanedithiol in CH.sub.2 Cl.sub.2 (1 time)                      3     50% trifluoroacetic acid (TFA) +                                                                       20.0                                                 5% 1,2-ethanedithiol in CH.sub.2 Cl.sub.2 (1 time)                      4     CH.sub.2 Cl.sub.2 wash (3 times)                                                                       0.5                                            5     CH.sub.3 OH wash (2 times)                                                                             0.5                                            6     10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                    0.5                                                  neutralization (2 times)                                                7     CH.sub.3 OH wash (2 times)                                                                             0.5                                            8     10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                    0.5                                                  neutralization (2 times)                                                9     CH.sub.3 OH wash (2 times)                                                                             0.5                                           10     CH.sub.2 Cl.sub.2 wash (2 times)                                                                       0.5                                           11     *Boc--amino acid (1 mmole/g resin)                                                                     120                                                  plus equivalent amount of                                                     dicyclohexylcarbodiimide (DCC) in                                             CH.sub.2 Cl.sub.2                                                      12     CH.sub.2 Cl.sub. 2 wash (1 time)                                                                       0.5                                           13     50% dimethylformamide in CH.sub.2 Cl.sub.2                                                             0.5                                                  wash (2 times)                                                         14     10% triethylamine (Et.sub.3 N) in CH.sub.2 Cl.sub.2                                                    0.5                                                  wash (1 time)                                                          15     CH.sub.3 OH wash (2 times)                                                                             0.5                                           16     CH.sub.2 Cl.sub.2 wash (2 times)                                                                       0.5                                           17     25% acetic anhydride in CH.sub.2 Cl.sub.2                                                              20.0                                                 (2 ml/g resin)                                                         18     CH.sub.2 Cl.sub.2 wash (2 times)                                                                       0.5                                           19     CH.sub.3 OH wash (2 times)                                                                             0.5                                           ______________________________________                                         *For the coupling of Asn and Gln,an 1.136 molar excess of                     1hydroxybenzotriazole (HOBt) was included in this step.                  

Briefly, for the coupling reaction, one mmol. of BOC-protected aminoacid in methylene chloride is used per gram of resin, plus oneequivalent of 0.5 molar DCCI in methylene chloride or 30% DMF inmethylene chloride, for two hours. When Arg is being coupled, a mixtureof 10% DMF and methylene chloride is used. Bzl is used as the hydroxylside-chain protecting group for Ser and Thr. 2-chloro-benzyloxycarbonyl(2Cl-Z) is used as the protecting group for the Lys side chain. Tos isused to protect the guanidino group of Arg, and the Glu or Asp carboxylgroup is protected as the Bzl ester. The phenolic hydroxyl group of Tyris protected with 2,6-dichlorobenzyl. At the end of the synthesis, thefollowing composition is obtained: X¹-Tyr(X²)-Ala-Asp(X³)-Ala-Ile-Phe-Thr(X⁴)-Asn-Ser(X⁵)Tyr(X²)-Arg(X⁶)-Lys(X⁷)-Ile-Leu-Gly-GlnLeu-Ser(X⁵)-Ala-Arg(X⁶)-Lys(X⁷)-Leu-Leu-Gln-Asp(X³)-Ile-Met-AsnArg(X⁶)-Gln-Gln-Gly-Glu(X³)-Arg(X⁶)-Asn-Gln-Glu(X.sup.3)-Gln-Gly-Ala-Lys(X⁷)-Val-Arg(X⁶)-Leu-X⁸

wherein X¹ is BOC, X² is 2,6-dichlorobenzyl, X³ is benyzl ester, X⁴ isBzl, X⁵ is Bzl, X⁶ is Tos, X⁷ is 2Cl-Z and X⁸ is -NH-MBHA resin support.

After the final Tyr residue has been coupled to the resin, the BOC groupis removed with 45% TFA in CH₂ Cl₂. In order to cleave and deprotect theremaining protected peptide-resin, it is treated with 1.5 ml. anisole,0.25 ml. methylethylsulfide and 10 ml. hydrogen fluoride (HF) per gramof peptide-resin, at -20° C. for one-half hour and at 0° C. for one-halfhour. After elimination of the HF under high vacuum, the resin-peptideremainder is washed alternately with dry diethyl ether and chloroform,and the peptide is then extracted with degassed 2N aqueous acetic acid.Lyophilization of the acetic acid extract provides a white fluffymaterial.

The cleaved and deprotected peptide is then dissolved in 30% acetic acidand subjected to Sephadex G-50 fine gel filtration.

The peptide is then further purified by CM-32 carboxymethyl cellulose(Whatman) cation-exchange chromatography(1.8×18 cm., V_(bed) =50 ml.)using a concave gradient generated by dropping 1 L. of 0.4 M NH₄ OAc, pH6.5 into a mixing flask containing 400 ml. 0.01 M. NH40Ac, pH 4.5. Finalpurification is carried out using partition chromatography on SephadexG-50 fine support (Pharmacia) with a nBuOH:EtOH:pyridine:0.2% N HOAc(4'1:1:7) solvent system. Purification details are generally set forthin Ling et al. Biochem. Biophys. Res. Commun. 95, 945 (1980). Thechromatographic fractions are carefully monitored by TLC, and only thefractions showing substantial purity are pooled.

The synthesis is repeated using a chloromethylated resin to produce thesame peptide having a free acid C-terminus, generally following theprocedure described in Biopolymers, 12, 2513-19 (1973) to link Leu tothe chloromethylated resin.

EXAMPLE II

The peptide oGRF(1-44) is isolated from ovine hypothalami extracts whicheffect secretion in vitro of immunoreactive growth hormone by ratpituitary cells following the procedure described in Biochem. Bioph.Res. Comm., Vol 116, 726-734 (1983) for the isolation of porcine andbovine hypothalamic GRFs, respectively. The starting material from whichthe peptide was derived was hypothalami from about 2100 sheep brainswhich were lyophilized. The hypothalami are boiled for two minutes, andafter cooling, an equal volume of 0.6 M HCl containing enzyme inhibitorswas added. They are then homogenized and centrifuged. The supernatant isdefatted with petroleum ether and diethyl ether, and the aqueous phaseis neutralized to pH 7.4. The salt concentration is then adjusted toabout that of saline by dilution, and the liquid is then pumped throughan Affi-Gel 10 (Bio Rad Laboratories) affinity column (7.2×3.5 cm.) thathas been coupled to antibodies raised against hGRF. After washing offthe unbound material, the adsorbed oGRF material is thereafter elutedfrom the column with 1 M acetic acid and then applied to a Sephadex G75column (4.5×120 cm.). It is then eluted with 1 M. acetic acid containing0.2% β-mercaptoethanol. The eluate fractions are assayed by testingtheir ability to release GH from rat pituitary cells in a monolayerculture as previously reported by Brazeau et. al. P.N.A.S., USA, 79,7909-7913 (1982) and/or by using a heterologous RIA with antisera raisedagainst hGRF(1-40). The bioreactive or immunoreactive fractions are thenpumped onto a reverse phase semipreparative C18 column. Elution fromthis column is effected with 0.25M TEAP in an acetonitrile gradient. Themajor bioreactive or immunoreactive fraction is then applied to ananalytical C18 column using a 0.2 vol % aqueous heptafluorobutyricacid/acetonitrile solvent system. Two bio- and immunoreactive fractionsfrom this column, if not already pure, are further run on an analyticalRP-HPLC C4 column using an 0.1% TFA/acetonitrile gradient to get twofractions of pure oGRF. The major fraction is tested as set forthhereinafter.

Amino acid analysis of the isolated peptide is carried out followinghydrolysis in sealed tubes using methodology as described in Anal.Biochem., 126, 144-156 (1982) using a Liquimat III amino acid analyzer,giving the following molar ratios: Asx(5), Thr(1), Ser(2), Glx(8),Gly(3), Ala(4), Val(1), Met(1), Ile(3), Leu(5), Tyr(2), Phe(1), Lys(3),and Arg(5). Sequencing of this isolated peptide from the major fractiongives the formula set forth hereinbefore.

The earlier eluting fraction has the same amino acid composition as themajor fraction oGRF peptide described hereabove and is probably itsmethionineoxidized form.

EXAMPLE III

To determine the effectiveness of the peptide to promote the release ofgrowth hormone, in vitro assays are carried out using synthetichGRF(1-44)-NH₂ in side-by-side comparison with equimolar concentrationsof the extracted and purified oGRF(1-44) of Example II. hGRF thus servesas a GRF Reference Standard having a known effectiveness to promote therelease of growth hormone from rat pituitary cells. Cultures are usedwhich include cells of rat pituitary glands removed some four to fivedays previously. Both cultures of a defined standard medium and cultureswhich are considered optimal for the secretion of growth hormone areused for the comparative testing, in the general manner described inBrazeau, et al. Regulatory Peptides, 1, 255, 1981. Incubation with thesubstance to be tested is carried out for 3 to 4 hours, and aliquots ofthe culture medium are removed and processed to measure their contentsin immunoreactive GH(ir GH) by a well-characterized radioimmunoassay.

The results of this comparative testing shows that, in equimolar ratios,the oGRF(1-44) has the full intrinsic biological activity of thesynthetic peptide and close to the same potency. In multiple dosesfactorial design experiments, oGRF is shown to have about the sameintrinsic activity as hGRF(1-44)-NH₂ and a specific activity equal toabout 70% of hGRF(1-44)-NH₂.

Approximately 1 out of 7000 to 15,000 children born in the USA are knownto be pituitary growth hormone deficient or "pituitary-dwarfs", i.e.,they are dwarfs because they lack the normal levels of pituitary GH intheir blood. There are clinical reasons to propose that most of thesepatients have a normal pituitary gland and that the cause of theirproblem is a lack either of the synthesis, or of the secretion, of thehypothalamic releasing factor for GH. Synthetic oGRF may be used totreat these cases who have heretofore been treated by injections ofhuman pituitary GH, an extremely expensive preparation obtainedexclusively from human pituitaries at autopsies. Human GH prepared byDNA-recombinant methodology, though announced in the literature, is notcurrently available for routine use.

Synthetic oGRF may also be used as a routine test for GH secretion incases in which a specific defect of pituitary function is suspected by aphysician. Synthetic oGRF may also replace the cumbersome methods usedcurrently (arginine infusions, hypoglycemia, L-DOPA injections, etc.) toassess GH secretory ability as a diagnostic procedure and may also beuseful for other purposes recently postulated for hGRF.

For administration to humans, synthetic oGRF peptides should have apurity of at least about 93% and preferably at least 98%. This puritymeans the intended peptide constitutes the stated weight % of all likepeptides and peptide fragments present. Most of the biologically activepeptides have been found to possess biological activities other thanthose for which they were originally recognized. In view of suchprecedents, it is likely that oGRF will be found to possessextrapituitary activities which may be of practical interest.

Chronic administration of synthetic oGRF peptides to farm animals,particularly sheep, or other warm-blooded animals is expected to promoteanabolism and thus increase body weight in terms of muscle mass. The usein veterinary medicine of the GRF of its species, i.e. oGRF in ovine, isthe ideal situation since the molecule injected or otherwiseadministered will not be antigenic, being of the same species as that ofthe animal treated. It should also increase milk production in ewes. Usein aquiculture for raising fish and other cold-blooded marine animals toaccelerate growth may also be important. Administration to animals at apurity as low as about 5% may be acceptable and will generally becarried out using a combination of the peptide and a veterinarilyacceptable solid or liquid carrier to form what for purposes of thisapplication is broadly termed a pharmaceutical composition.

Synthetic oGRF or the nontoxic salts thereof, combined with apharmaceutically acceptable carrier to form a pharmaceuticalcomposition, may be administered to mammals, including humans, eitherintravenously, subcutaneously, intramuscularly, intranasally or orally.The administration may be employed by a physician to stimulate therelease of growth hormone where the host being treated requires suchtherapeutic treatment. The required dosage will vary with the particularcondition being treated, with the severity of the condition and with theduration of desired treatment.

Such peptides are often administered in the form of pharmaceuticallyacceptable nontoxic salts, such as acid addition salts or metalcomplexes, e.g., with zinc, iron or the like (which are considered assalts for purposes of this application). Illustrative of such acidaddition salts are hydrochloride, hydrobromide, sulphate, phosphate,maleate, acetate, citrate, benzoate, succinate, malate, ascorbate,tartrate and the like. If the active ingredient is to be administered intablet form, the tablet may contain a binder, such as tragacanth, cornstarch or gelatin; a disintegrating agent, such as alginic acid; and alubricant, such as magnesium stearate. If administration in liquid formis desired, sweetening and/or flavoring may be used, and intravenousadministration in isotonic saline, phosphate buffer solutions or thelike may be effected.

The peptides should be administered under the guidance of a physician,and pharmaceutical compositions will usually contain the peptide inconjunction with a conventional, pharmaceutically-acceptable carrier.Usually, the dosage will be from about 20 to about 2000 nanograms of thepeptide per kilogram of the body weight of the host.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto. For example,modifications in the 44-member chain, particularly deletions beginningat the carboxyl terminal of the peptide, can be made in accordance withthe known experimental evidence previously obtained with hGRF andfollowing the practises to date to create fragments 34 to 43 residues inlength, e.g. oGRF(1-40) and oGRF(1-37), or even shorter fragments, i.e.oGRF(1-32), which fragments may have either NH₂ or OH at the C-terminalthat retain the intrinsic biological activity of the peptide, and suchshorter peptides are considered as being within the scope of theinvention. Moreover, additions can be made to either terminal, or toboth terminals, and/or generally equivalent residues can be substitutedfor naturally occurring residues, as is well-known in the overall art ofpeptide chemistry to produce analogs having at least a substantialportion of the potency of the native polypeptide without deviating fromthe scope of the invention.

Particular features of the invention are set forth in the claims whichfollow.

What is claimed is:
 1. A peptide having the formula:H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-YwhereinY is OH or NH₂ or a nontoxic salt of said peptide.
 2. A syntheticpeptide having the formula of claim 1 wherein Y is NH₂.
 3. A syntheticpeptide having the formula of claim 1 wherein Y is OH.
 4. A compositionfor accelerating the growth of sheep comprising an effective amount of apeptide as defined in claim 2 and a veterinarily acceptable solid orliquid carrier therefor.
 5. A composition for use in accelerating thegrowth of sheep comprising an effective amount of a peptide having thesequence:Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-AIa-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leuor a nontoxic salt of said peptide, plus a veterinarily acceptable solidor liquid carrier therefor.
 6. A method of accelerating the growth ofsheep comprising administering an effective amount of a peptide-havingthe sequence:Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu,or a nontoxic salt of said peptide.
 7. A method in accordance with claim6 wherein the carboxyl terminus is free acid.
 8. A method in accordancewith claim 6 wherein the carboxyl terminus is amide.
 9. A method ofstimulating the release of GH in sheep by administering intravenously,subcutaneously, intramuscularly, intranasally or orally about 20 toabout 2000 nanograms of the peptide of claim 1 per kilogram of bodyweight.
 10. A method of stimulating the release of GH in sheep byadministering intravenously, subcutaneously, intramuscularly,intranasally or orally about 20 to about 2000 nanograms of the peptideof claim 2 per kilogram of body weight.
 11. A method of stimulating therelease of GH in sheep by administering intravenously, subcutaneously,intramuscularly, intranasally or orally about 20 to about 2000 nanogramsof the peptide of claim 3 per kilogram of body weight.
 12. A method ofincreasing muscle . mass in sheep by administering intravenously,subcutaneously, intramuscularly, intranasally or orally about 20 toabout 2000 nanograms of the Peptide of claim 1 per kilogram of bodyweight.